The Escherichia coli LTR-7TAR, Bj, rec B-, has been deposited on Jan. 17, 1990 in the American Type Culture Collection (ATCC), 10801 University Blvd, Manassas, Va. 20110 under the accession number 68203.
1. Field of the Invention
The present invention relates to a method of effecting viral inhibition with DNA sequences encoding multiple target response elements, and to constructs suitable for use in same. In particular, the invention relates to a method of inhibiting replication of the Human Immunodeficiency Virus (HIV).
2. Background Information
The tat protein of HIV transactivates viral gene expression and is essential for production of viral products (Arya et al., Science 229:69-73 (1985); Sodroski, et al., Science 229:74-77 (1985); Dayton, et al., Cell 44:941-947 (1986); Fisher, et al., Nature 320:367-371 (1986)). The tat activation response element (TAR) has been localized within the region of the first 44 nucleotides downstream of the transcription initiation site (Chen and Okayama, Mol. Cell. Biol. 7:2745 (1987); Rosen, et al., Cell 41:813-823 (1985); Tong-Starksen, et al., Proc. Natl. Acad. Sci., USA 84:6845-6849 (1987); Haruber, et al., J. Virol. 62:673-679 (1988)). This region, present in all HIV-1 transcripts, forms an unusually stable stem loop structure (Okamoto and Wong-Staal, Cell 47:29-35 (1986)), and several lines of evidence suggest that the transcriptional effect of tat is mediated through interaction with the TAR region of viral RNA (Sharp, et al. Cell 59:229-230 (1989); Viscidi, et al., Science 246:1606-1608 (1989); Berkhout, et al., Cell 59:273-282 (1989); Garcia, et al., EMBO J. 8:765-778 (1989); Feng and Holland, Nature 334:165-167 (1988); Southgate, et al., Nature 345:640-642 (1990)).
While tat binding to TAR RNA sequences has been demonstrated (Rappaport, et al., Cold Spring Harbor, N.Y. (1989b); Dingwall, et al., Proc. Natl. Acad. Sci. USA 86:6925-6929 (1989)), the sequence requirements for tat binding are not sufficient to explain the sequence and structural requirements needed for transactivation. Cellular factors also appear to play a role in tat mediated transactivation which may confer additional specificity (Marciniak, et al., Proc. Natl. Acad. Sci. 87:3624-3628 (1990)). Tat appears to function poorly in nonprimate cells and studies using interspecific hybrids suggest that transactivation potential is correlated with the presence of human chromosome 12 (Hart, et al., Science 246:488-491). Several cellular TAR RNA as well as TAR DNA binding proteins have been identified (Gaynor, R. B. EMBO J. 8:765-778 (1989); Gatignol, et al., Proc. Natl. Acad. Sci. USA 86:7828-7832 (1989); Wu, et al., EMBO J. 7:2117-2129 (1988); Jones, et al., Science 232:755-758 (1986); Garcia, et al., EMBO J. 8:765-778 (1989); Marciniak, et al., Proc. Natl. Acad. Sci. 87:3624-3628 (1990)), although the role of these proteins in tat mediated transactivation is unclear.
In vitro, tat protein can be released and taken up by cells (Frankel and Pabo, Cell 55:1189-1193 (1988)), and has biological effects on the regulation of cellular proliferation in addition to its role in HIV promoter activation. Recent studies indicate that tat inhibits antigen-induced lymphocyte proliferation, (Viscidi, et al., Science 246:1606-1608 (1989)), has growth promoting activity on cells derived from Kaposi Sarcoma lesions of AIDS patients (Ensoli, et al., Nature 340:84-86 (1990)) and stimulates the production of inflammatory cytokines (Buonaguro et al., (1992) J. Virol., 66:7159-7167). In contrast, tat does not cause significant reduction of lymphocyte proliferation in response to mitogens. Since the tat protein is critical to HIV replication and the onset of AIDS, interference with tat function will be therapeutically significant.
Transdominant mutations of HIV proteins have been reported (Malim, et al., Cell 58:205-214 (1989); Torno, et al., Cell 59:113-120 (1989); Marciniak, et al., Proc. Natl. Acad. Sci. 87:3624-3628 (1990). These proteins, produced constitutively from a strong promoter, can antagonize the growth of HIV-1 and, therefore, can be used to create cell lines "immunized" to viral infection.
Since TAR RNA appears to interact with tat protein directly (Southgate, et al., Nature 345:640-624 (1990)) or through the combined activities of cellular factor(s) (Marciniak, et al., Proc. Natl. Acad. Sci. USA 87:3624-3628 (1990), we hypothesized that TAR RNA, produced in large amounts, might serve as a competitive inhibitor of tat function.